The invention is directed to a process for providing an assay protocol that measures enzymatic activity. More particularly, the invention is directed to a process to precisely and conveniently quantitate enzymatic activity of protein kinases and further to provide an assay specific for individual protein kinases in the presence of other protein kinases.
A bibliography of the references cited in this application can be found in the section preceding the claims.
Enzymes are large proteins that catalyze reactions in living cells. Enzymes build up or tear down other molecules. For example, enzymes catalyze the synthesis of fat from fatty acids, form complex sugars from glucose and fructose, and aid in the formation of other proteins from amino acids. Enzymes also reverse the build-up process by breaking down more complex structures. Enzymes are generally specific to certain substrates for their reactions. For example, an individual enzyme may catalyze the reaction where only one substrate is involved or it may act on a group of related substrates.
In healthy persons, most enzymes are found within cells. Some diseases however cause the release of enzymes from dying cells into the blood. The increased levels of enzymes can then be measured. An abnormal level of enzymes in the blood characterizes certain medical conditions. For example, an enzyme assay for abnormal levels of the enzyme creatine kinase in the blood is useful as a diagnostic measure of heart disease. In like manner, bone or liver diseases can be diagnosed by observing increasing levels of alkaline phosphatase in the blood stream. Prostate cancer is diagnosed by increased levels of acid phosphatase in the blood stream.
Enzymes are classified into groups according to the general kind of reaction they catalyze. The present invention refers to a specific group of enzymes called transferases, which catalyze the transfer of a group from one substrate to another. The present invention is specifically directed to the transferase subgroup called protein kinases.
Protein kinase is a generic name for all enzymes that transfer a phosphate to a protein. About three to four percent of the human genome contains transcription information for the formation of protein kinases. Currently, there are up to 200 known different protein kinases. However, because three to four percent of the human genome is a code for the formation of protein kinases, there may be many thousands of distinct and separate kinases in the human body.
Protein kinases are enzymes which catalyze the transfer of phosphorous from adenosine triphosphate (ATP), or guanosine triphosphate (GTP) to the targeted protein to yield a phosphorylated protein and adenosine diphosphate (ADP) or guanosine diphosphate (GDP), respectively. ATP or GTP is first hydrolyzed to form ADP or GDP and inorganic phosphate. The inorganic phosphate is then attached to the targeted protein. The protein substrate which is targeted by kinases may be a structural protein, found in membrane material such as a cell wall, or another enzyme which is a functional protein.
Due to their physiological relevance, variety and ubiquitousness, protein kinases have become one of the most important and widely studied family of enzymes in biochemical and medical research. Studies have shown that protein kinases are key regulators of many cell functions, including signal transduction, transcriptional regulation, cell motility, and cell division. Several oncogenes have also been shown to encode protein kinases, suggesting that kinases play a role in oncogenesis.
Protein kinases are often divided into two groups based on the amino acid residue they phosphorylate. The first group, called serine/threonine kinases, includes cyclic AMP and cyclic GMP dependent protein kinases, calcium and phospholipid dependent protein kinase, calcium and calmodulin-dependent protein kinases, casein kinases, cell division cycle protein kinases and others. These kinases are usually cytoplasmic or associated with the particulate fractions of cells, possibly by anchoring proteins.
The second group of kinases, called tyrosine kinases, phosphorylate tyrosine residues. They are present in much smaller quantities but play an equally important role in cell regulation. These kinases include several receptors for molecules such as growth factors and hormones, including epidermal growth factor receptor, insulin receptor, platelet derived growth factor receptor and others. Studies have indicated that many tyrosine kinases are transmembrane proteins with their receptor domains located on the outside of the cell and their kinase domains on the inside.
Phosphorylation of serine-, threonine- and tyrosine-containing proteins by kinases is important because the phosphorylated protein products have been implicated in a variety of cellular processes including oncogenesis, cellular transformation, cellular growth and exocytosis. Currently, much experimentation is performed involving kinases which may inhibit cancer growth or promote cancer cell death. Determining the specific kinase involved in inhibiting cancer growth or promoting cell death is important to society. Therefore, advances in recognizing kinase activity levels are extremely important.
Activity Determination
Reference is made to Robyt and White (1990) which is incorporated herein by reference, for a general description of methods for determining the activity of an enzyme. Robyt and White defines the activity of an enzyme as the amount of reaction that a certain amount of enzyme will produce in a specified period of time. The activity is determined by measuring the amount of product produced or the amount of substrate used up per unit of time under high concentrations or saturating conditions of substrate. This is usually accomplished by performing a chemical analysis for the product or substrate.
Substrates that are typically used in an assay for specific kinase activity include casein, which is isolated from milk; histones, isolated from calves; phosphovitin, isolated from egg yolks; and myelin basic proteins, isolated from bovine spinal cords. These substrates can be phosphorylated in an assay, assuming that the correct kinase has been chosen. Assays utilizing these substrates to determine kinase activity are well known in the prior art.
Radioactive Detection of Kinase Activity
Most current methods of measuring protein kinase activity are based on the radioactive detection method. In these methods, a sample containing the kinase of interest is incubated with activators and a substrate in the presence of xcfx84-32P-ATP or xcfx84-32P-GTP. Often, a general and inexpensive substrate such as histone or casein is used. After a suitable incubation period, the reaction is stopped and an aliquot of the reaction mixture is placed directly onto a filter which binds the substrate. The filter is then washed several times to remove excess radioactively-labeled free ATP, and the amount of radio-labeled phosphate incorporated into the substrate is measured by scintillation counting. This method is widely used and provides an accurate method for determining protein kinase activity in both crude and purified samples.
Babcook et al. (1991) also describe an assay using monoclonal antibodies and immunofluorescence technology for the determination of protein-tyrosine kinase and protein-tyrosine phosphatase activities. The method was performed utilizing p56lck or p60src.
Budde et al. (1991) disclose assay techniques utilizing acidic peptide substrates of protein kinases. This technology uses radioactive phosphorous placed in a substrate to be studied. After kinase activity, the phosphopeptide is eluted while the individual radioactive phosphorus, ATP and protein are impeded.
Gopalakrishna et al. (1992) disclose a method which utilizes the conventional approaches to measure protein kinase activity. The method combines the incubations and filtrations necessary to determine the protein kinase activity using multi-well plates with fitted filtration disks. In a related reference by Chakravarthy et al. (1990), kinase C activity is measured by using protein kinase C (PKC) selective peptide substrates by incorporating radioactive phosphorous into the substrate. The radioactivity is measured by liquid scintillation.
Non-Radioactive Determination of Kinase Activity
A non-radioactive method of detecting kinase activity has been developed in which tyrosine phosphorylation is detected by using anti-phosphotyrosine antibodies (Rijksen et al., 1991). After incubation of the tyrosine kinase with unlabeled ATP and a suitable substrate, the reaction mixture is subjected to a dot blot assay on a polyvinylidene diflouride (PVDF) membrane. The extent of phosphorylation is determined by reaction with anti-phosphotyrosine antibody, followed by detection with an immunogold staining procedure. The amount of phosphotyrosine present is detected with a densitometer.
A disadvantage of the dot blot method is that it is limited to detecting tyrosine kinases. Antibodies to phosphotyrosine can be produced due to the size of the antigen. Attempts to produce similar antibodies to phosphoserine and phosphothreonine have not been successfully employed to assay for phosphoserine- and phosphothreonine-containing proteins. In addition, the assay requires several incubation and washing steps, each taking a considerable amount of time, which results in a greatly extended assay completion time. The result of the assay is a colored dot on the blot. The colored dot may limit the effective sample range of the assay and may require the user to quantitate the final results using a scanning densitometer, which is an expensive piece of equipment not available in all laboratories. The densitometer must have beam dimensions covering at least the targeted cross section of the dots generated.
The present invention provides for a method of determining the presence of activity of a selected protein kinase, comprising conjugating a binding compound to a peptide substrate forming a modified peptide substrate; adding a sufficient quantity of the modified peptide substrate to a solution containing the selected protein kinase; incubating the protein kinase with the modified peptide substrate under conditions where the protein kinase is active for a time sufficient to form a modified peptide product; and measuring the activity of the protein kinase.
The present invention is further directed to a kit for determining the presence or activity of a selected protein kinase comprising a container containing a modified peptide substrate having specific reactivity to the protein kinase and modified by chemical reaction to allow quantitation; and instructions for use.
The present invention is also directed to a kit for determining the presence or activity of a tyrosine kinase comprising a container containing a modified peptide substrate selected from the group consisting of biotinylated Promega peptide G (SEQ. ID. 7) and analogs and combinations thereof; a container containing a biotin-binding matrix; and instructions for use.
The present invention is also directed to a kit for determining the presence or activity of serine-threonine kinases comprising a container holding a modified peptide substrate selected from the group consisting of biotinylated Promega peptide A (SEQ. ID. 1), biotinylated Promega peptide B (SEQ. ID. 2), biotinylated Promega peptide C (SEQ. ID. 3), biotinylated Promega peptide D (SEQ. ID. 4), biotinylated Promega peptide E (SEQ. ID. 5), biotinylated Promega peptide F (SEQ. ID. 6), biotinylated Promega peptide G (SEQ. ID. 7), biotinylated Promega peptide H (SEQ. ID. 8) and analogs and combinations thereof; a container holding a biotin-binding matrix; and instructions for use.
Further, the present invention is directed to a method for detecting the presence or activity of a selected protein kinase in a body fluid comprising reacting the body fluid with a sufficient amount of a biotinylated peptide substrate forming a modified peptide product under conditions where the protein kinase is active for a time sufficient to form the modified peptide product in an amount such that the modified peptide product may be detected and measuring the amount of modified peptide product.
The present invention makes it possible to assay for a specific protein kinase in the presence of several other proteins kinases in a tissue extract. This type of assay is highly desirable to investigators because the purification of the specific protein kinase under study is not required. Conventionally, purification is required to remove other protein kinases in the extract. This lengthy approach may be circumvented by using the specific biotinylated peptide substrate approach.
In addition, the investigator is aided in determining the expression level of the specific kinase under various physiological conditions with minimal losses in the enzyme, since purification is obviated. This means that the activity assayed is an accurate estimate of the total active enzyme expressed.
Peptide substrates for several protein kinases such as cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), Ca2+/phospholipid-dependent protein kinases (PKC), casein kinases 1 and 2 (CK-1 and CK-2), growth factor receptors, non-growth factor receptors and soluble proteins that contain an active tyrosine kinase, cell cycle-dependent protein kinase (p34cdc2 protein kinase), S6 protein kinase, Ca2+/calmodulin-dependent or multifunctional (CAM) protein kinases, DNA-dependent protein kinase, and carboxyl-terminal domain (CDT) kinases are custom synthesized using a peptide synthesis protocol that incorporates a biotinylated amino acid at the N-terminus of the peptide. Thus, there are several protein kinases that can be assayed by the present procedure providing for the ability to make several kits for use with the various protein kinases.
The assay is very fast and may typically be completed in less than 10 minutes after termination of the reaction. This is important since current protocols require approximately 2 hours to obtain the same objectives.
No special equipment is required other than standard equipment that is currently available in most laboratories. The procedure does not require special inorganic or organic solvents such as phosphoric acid, acetic acid, acetone or ethanol, found in prior art protocols.
The present invention is amenable to be adapted to a large scale assay which may be demanded by large laboratories or pharmaceutical laboratories engaged in drug research.
The cost of the present invention is similar to the cost of prior art assays. The projected increased cost of avidin and biotinylating reagent may be offset by the elimination of expensive solvents required by other assays.
Further, the present invention eliminates the need for the use of an assay system requiring gel electrophoresis to separate the exogenous peptide from endogenous substrates. Gel electrophoresis separates the various mono- and phosphorylated forms of the substrate that may be useful. However, this method further complicates the quantitation of total phosphate transfer.
Problems are often encountered when utilizing the prior art filter binding paper assay to bind strongly positive molecules that are present in the tissue of cellular extract and are phosphorylated. The present invention overcomes the problems because only the substrate and product are bound since they are the only components that are biotinylated.
The present invention eliminates the need to change the primary structure of the consensus sequence, which is the primary amino acid sequence target of the enzyme being analyzed, by adding additional arginine residues in order to maintain the primary sequence for the enzyme that is present in vivo. Prior art assays utilizing filter binding require the substrates to contain multiple arginine residues which may alter the specificity of the substrate such that the substrate may become suitable for another kinase. An example of this alteration is given by the sequence Arg-Arg-Arg-Tyr-Ser (SEQ. ID. 9) (a key to amino acid abbreviations is found in Table 1) that is present in the CDT kinase peptide substrate Arg-Arg-Arg(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 (SEQ. ID. 10) which might be recognized by cAMP-dependent protein kinase and other kinases recognizing this sequence.
The present invention yields results with a greater probability of accuracy since virtually all of the phosphorylated peptide molecules are captured by the strong binding avidin or streptavidin-coated matrix. Conversely, the phosphorylated proteins aliquoted onto the phosphocellulous paper are bound by an electrostatic interaction that is not strong. Therefore, loss of phosphorylated peptide occurs during the washing procedure.
Reference is now made in detail to the preferred embodiments of the invention, examples of which are illustrated in the accompanying figures.